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1.
A comparison of the photoregulation of development has been made for etiolated and light-grown plants of wild-type (WT) tobacco (Nicotiana tabacun L.) and an isogenic transgenic line which expresses an introduced oat phytochrome gene (phyA) under the control of a constitutive viral promoter. Etiolated seedlings of both the WT and transgenic line showed irradiance-dependent inhibition of hypocotyl growth under continuous far-red (FR) light; transgenic seedlings showed a greater level of inhibition under a given fluence rate and this is considered to be the result of the heterologous phytochrome protein (PhyA) functioning in a compatible manner with the native etiolated phytochrome. Deetiolation of WT seedlings resulted in a loss of responsiveness to prolonged FR. Light-grown transgenic seedlings, however, continued to respond in an irradiance-dependent manner to prolonged FR and it is proposed that this is a specific function of the constitutive PhyA. Mature green plants of the WT and transgenic lines showed a qualitatively similar growth promotion to a brief end-of-day FR-treatment but this response was abolished in the transgenic plants under prolonged irradiation by this same FR source. Growth inhibition (McCormac et al. 1991, Planta 185, 162–170) and enhanced levels of nitrate-reductase activity under irradiance of low red:far-red ratio, as achieved by the FR-supplementation of white light, emphasised that the introduced PhyA was eliciting an aberrant mode of photoresponse compared with the normal phytochrome population of light-grown plants. Total levels of the oat-encoded phytochrome in the etiolated transgenic tobacco were shown to be influenced by the wavelength of continuous irradiation in a manner which was qualitatively similar to that seen for the native, etiolated tobacco phytochrome, and distinct from that seen in etiolated oat tissues. These results are discussed in terms of the proposal that the constitutive oat-PhyA pool in the transgenic plants leads to a persistence of a mode of response normally restricted to the situation in etiolated plants.Abbreviations FR
far-red light
- R
red light
- WL
white light
- WL + FR
white light supplemented with FR
- HIR
high-irradiance response
- PAR
photosynthetically active radiation
- Pr, Pfr
R- and FR-absorbing forms of phytochrome
- Ptot
total phytochrome
-
phyA (PhyA)
gene (encoded protein) for phytochrome
- WT
wild type
This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.M.; J.R. Cherry and R.D. Vierstra, (Department of Horticulture, University of Wisconsin-Madison, USA) are thanked for the provision of the transgenic tobacco line. 相似文献
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Use of the GFP reporter as a vital marker for Agrobacterium-mediated transformation of sugar beet (Beta vulgaris L.) 总被引:4,自引:0,他引:4
Zhang CL Chen DF McCormac AC Scott NW Elliott MC Slater A 《Molecular biotechnology》2001,17(2):109-117
Molecular approaches to sugar beet improvement will benefit from an efficient transformation procedure that does not rely
upon exploitation of selectable marker genes such as those which confer antibiotic or herbicide resistance upon the transgenic
plants. The expression of the green fluorescent protein (GFP) signal has been investigated during a program of research that
was designed to address the need to increase the speed and efficiency of selection of sugar beet transformants. It was envisaged
that the GFP reporter could be used initially as a supplement to current selection regimes in order to help eliminate “escapes”
and perhaps eventually as a replacement marker in order to avoid the public disquiet associated with antibiotic/herbicide-resistance
genes in field-released crops. The sgfp-S65T gene has been modified to have a plant-compatible codon usage, and a serine to threonine mutation at position 65 for
enhanced fluorescence under blue light. This gene, under the control of the CaMV 35S promoter, was introduced into sugar beet
via Agrobacterium-mediated transformation. Early gene expression in cocultivated sugar beet cultures was signified by green fluorescence several
days after cocultivation. Stably transformed calli, which showed green fluorescence at a range of densities, were obtained
at frequencies of 3–11% after transferring the inoculated cultures to selection media. Cocultivated shoot explants or embryogenic
calli were regularly monitored under the microscope with blue light when they were transferred to media without selective
agents. Green fluorescent shoots were obtained at frequencies of 2–5%. It was concluded that the sgfp-S65T gene can be used as a vital marker for noninvasive screening of cells and shoots for transformation, and that it has
potential for the development of selectable marker-free transgenic sugar beet. 相似文献
4.
Manoj K. Mishra Santosini Devi Alex McCormac Nigel Scott DongFang Chen Malcolm Elliott Adrian Slater 《Biologia》2010,65(4):639-646
The green fluorescent protein (GFP) was used as a visual selectable marker to produce transgenic coffee (Coffea canephora) plants following Agrobacterium-mediated transformation. The binary vector pBECKS 2000.7 containing synthetic gene for GFP (sgfp) S65T and the hygromycin phosphotransferase gene hph both controlled by 35S cauliflower mosaic virus CaMV35S promoters was used for transformation. Embryogenic cultures were initiated from hypocotyls and cotyledon leaves of in vitro grown seedlings and used as target material. Selection of transformed tissue was carried out using GFP visual selection as
the sole screen or in combination with a low level of antibiotics (hygromycin 10 mg/L), and the efficiency was compared with
antibiotics selection alone (hygromycin 30 mg/L). GFP selection reduced the time for transformed somatic embryos formation
from 18 weeks on a hygromycin (30 mg/L) antibiotics containing medium to 8 weeks. Moreover, visual selection of GFP combined
with low level of antibiotics selection improved the transformation efficiency and increased the number of transformed coffee
plants compared to selection in the presence of antibiotics. Molecular analysis confirmed the presence of the sgfp-S65T coding region in the regenerated plants. Visual screening of transformed cells using GFP by Agrobacterium-mediated transformation techniques was found to be efficient and therefore has the potential for development of selectable
marker-free transgenic coffee plants. 相似文献
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A series of binary T-DNA vectors (pBECKS) has been created for use in theAgrobacterium-mediated genetic transformation of plants. The pBECKS series has corrected the undesirable features of the popular pBIN19
vector; the deleterious mutation within the coding sequence ofnptII has been amended and the cloning sites are now adjacent to the right border repeat in order to reduce the possibility of
producing truncated sequences of novel genes within transformants. One set of vectors incorporates various combiantions of
the marker genesgusA,C1/Lc,nptII,hph, andbar, for pursuit of early and stable transformation events. A set of constructs which contain deleted T-DNA borders in various
combinations and display predictably altered efficacies for gene transfer has also been created. A modular set of vectors
has been designed to facilitate the insertion and transfer of novel gene sequences by providing anptII-linked plant expression cassette orlacZ-multiple cloning site. A range of antibiotic resistance genes has been incorporated into the non-T-DNA part of the vectors
in order to facilitate their selection across the range ofAgrobacterium virulence strains. 相似文献
7.
Alex C. McCormac Joel R. Cherry Howard P. Hershey Richard D. Vierstra Harry Smith 《Planta》1991,185(2):162-170
The physiological responses of transgenic tobacco (Nicotiana tabacum L.) plants that express high levels of an introduced oat (Avena sativa L.) phytochrome (phyA) gene to various light treatments are compared with those of wild-type (WT) plants. Seeds, etiolated seedlings, and light-grown plants from a homozygous transgenic tobacco line (9A4) constructed by Keller et al. (EMBO J, 8, 1005–1012, 1989) were treated with red (R), far-red (FR), or white light (WL) with or without supplemental FR light, revealing major perturbations of the normal photobiological responses. White light stimulated germination of both WT and transgenic seed, but addition of FR to the WL treatment suppressed germination. In the WT, all fluence rates tested inhibited germination, but in the transgenics, reduction effluence rate partially relieved germination from the FR-mediated inhibition. It is suggested that the higher absolute levels of the FR-absorbing form of phytochrome (Pfr) in the irradiated transgenics, compared to the WT, may be responsible for the reduced FR-mediated inhibition of germination in the former. Hypocotyl extension of dark-grown seedlings of both WT and transgenic lines was inhibited by continuous R or FR irradiation, typical of the high-irradiance response (HIR). After 2 d of de-etiolation in WL, the WT seedlings had lost the FR-mediated inhibition of hypocotyl extension, whereas it was retained in the transgenics. The FR-mediated inhibition of hypocotyl extension in the transgenic seedlings after de-etiolation may reflect the persistence of an, FR-HIR response mediated by the overexpressed oat PhyA phytochrome. Light-grown WT seedlings exhibited typical shade-avoidance responses when treated with WL supplemented with high levels of FR radiation. Internode and petiole extension rates were markedly increased, and the chlorophyll ab ratio decreased, in the low-R: FR treatment. The transgenics, however, showed no increases in extension growth under low-R: FR treatments, and at low fluence rates both internode and petiole extension rates were significantly decreased by low R FR. Interpretation of these data is difficult. The depression of the chlorophyll ab ratio by low R FR was identical in WT and transgenic plants, indicating that not all shade-avoidance responses of light-grown plants were disrupted by the over-expression of the introduced oat phyA gene. The results are discussed in relation to the proposal that different members of the phytochrome family may have different physiological roles.Abbreviations FR
far-red light
- PAR
photosynthetically active radiation
- Pr, Pfr
red- and FR-absorbing forms of phytochrome
- Ptot
total phytochrome
-
PhyA (PhyA)
gene (encoded protein) for phytochrome
- R
red light
- WL
white light
- WT
wild type
This work was supported by an Agricultural and Food Research Council research grant to H.S. and A.C.M.; the production of the transgenic seed was funded by the U.S. Department of Energy (DE-F602-88ER13968) to R.D.V., and by E.I. du Pont de Nemours; Dr. G.C. Whitelam is thanked for the provision of monoclonal antibodies for the immunoblot analyses. 相似文献
8.
Light-signalling pathways leading to the co-ordinated expression of HEMA1 and Lhcb during chloroplast development in Arabidopsis thaliana 总被引:2,自引:0,他引:2
During de-etiolation, the co-ordinated synthesis of chlorophyll and the chlorophyll a/b-binding proteins is critical to the development of functional light-harvesting complexes. To understand how this co-ordination is achieved, we have made a detailed study of the light-regulated signalling pathways mediating the expression of the HEMA1 and Lhcb genes encoding glutamyl-tRNA reductase, the first committed enzyme of 5-aminolaevulinic acid formation, and chlorophyll a/b-binding proteins, respectively. To do this, we have screened 7 photoreceptor and 12 light-signalling mutants of Arabidopsis thaliana L. for induction of HEMA1 and Lhcb expression in continuous red, far-red and blue light and following a red pulse. We have categorised these mutants into two groups. The phyA, phyB, phyAphyB, cry1, cry2, cop1, det1, poc1, eid1, and far1 mutations lead to diverse effects on the light regulation of HEMA1, but affect Lhcb expression to a similar degree. The hy1, hy2, hy5, fin219, fhy1, fhy3, spa1, ndpk2, and pat1 mutants also affect light regulation of both HEMA1 and Lhcb expression, but with differences in the relative magnitude of the two responses. The fhy1 and fhy3 mutants show the most significant differences in light regulation between the two genes, with both showing a strong inhibition of HEMA1 expression under continuous red light. These results demonstrate that co-ordinated regulation of HEMA1 and Lhcb is largely achieved through parallel light regulation mediated by shared phytochrome- and cryptochrome-signalling pathways. However, glutamyl-tRNA reductase is also required for the synthesis of other tetrapyrroles and this dual role may account for the observed differences in these light-signalling pathways. 相似文献
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10.
Photoinduction and photoinhibition of germination in seed from a homozygous tobacco (Nicotiana tabacum L.) line containing an introduced oat phyA cDNA (encoding phytochrome A) is compared with that of isogenic wild-type (WT) tobacco. Under continuous irradiation by a light source with a low redfar-red (RFR) ratio the transgenic tobacco seed appeared to be less susceptible to photoinhibition of germination compared with WT seed. However, induction of germination following a short pulse by R (666 nm) was not enhanced in the genotype transformed by oat phyA cDNA compared with the WT; neither did germination of the transgenic tobacco seed show an increased sensitivity to saturating pulses of light of longer wavelengths (666–730 nm). In seeds of transgenic Arabidopsis thaliana (L.) Heynh. which contained an introduced phytochrome-B-encoding cDNA, levels of dark germination were enhanced, consistent with mediation of response by phytochrome B-Pfr. The germination behaviour of Arabidopsis genotypes wich contained an introduced cDNA encoding phytochrome A, however, did not significantly differ from that of the WT.Abbreviations ABO
seed transformed with Arabidopsis phyB
- cDNA; CaMV
cauliflower mosaic virus
- FR
far-red light
- Pfr
far-red-absorbing form of phytochrome
- Ptot
total phytochrome
- Pfr/Ptot
phytochrome photoequilibrium
- R
red light
- RBO
seed transformed with rice phyB cDNA
- RFR
quantum ratio of red and far-red light
- WL
white light
- WL + FR
whitelight supplemented with far-red light
- WT
wild type
The authors wish to thank R.D. Vierstra (Department of Horticulture, University of Wisconsin-Madison, USA) for providing the transgenic tobacco line, and M.T. Boylan, D. Wagner and P.H. Quail (U.C. Berkeley/USDA Plant Gene Expression Center, Albany, Calif. USA) for providing the transgenic Arabidopsis lines. The work presented in this paper was funded by grants from the Agricultural and Food Research Council (H.S., A.C.M., G.C.W.). 相似文献